Protein disulfide isomerase (PDI) has a key role in maintaining cellular homeostasis by mediating oxidative protein folding. It catalyzes disulfide bond formation, breakage and rearrangement in the endoplasmic reticulum (ER), and possesses chaperone protein activity. Increasing evidence suggests that PDI supports the survival and progression of several cancers and most significantly brain cancer. Cancer cells require higher levels of PDI to cope with significant ER stress and global increase in protein synthesis to sustain rapid proliferation. Increased protein synthesis leads to an abundant presence of misfolded proteins in the ER that need to be refolded by PDI. As such, cancer cells are more vulnerable to PDI inhibition than normal cells making PDI a new and exciting target to treat brain cancer. Although PDI remains a very promising oncology target, currently there are no PDI inhibitors under clinical development. There is an urgent need to develop drugs targeting essential pathways in brain cancer. Previous attempts at targeted therapy for glioma did not produced cures. Since PDI is a hub for orchestrating an entire process essential for rapid proliferation, selective blockade of its function will result in increased ER stress leading to the death of cancer cells. This approach is drastically different from previous attempts that target a single protein leading to facile escape and tumor recurrence. Previously, we discovered a class of novel and irreversible PDI inhibitors that selectively bind to PDI and demonstrated significant in vivo efficacy with no apparent systemic toxicity. More recently, we performed a high throughput screen and have identified several nM inhibitors of PDI representing the most potent PDI inhibitors discovered to date. We propose to optimize and characterize these compounds to select clinical leads for the treatment of glioblastoma multiforme (GBM). To determine the significance of PDI signaling in cancer progression and the effect of its inhibition on tumor growth, we propose the following aims. Aim 1: To validate PDI as a target and to determine the expression levels of PDI and select ER stress genes in GBM cell lines. Aim 2: To perform a structure-based and ADMET-guided lead optimization campaign to select the top 5 compounds with desirable potency, selectivity, and PK properties. Aim 3: To perform mechanistic studies of top 5 compounds from Aim 2 as single agents and in combination with temozolomide and radiation (TMZ/IR) using Bru-Seq technology in a panel of GBM cell lines. Aim 4: To determine in vivo efficacy of PDI inhibitors, as a single agent and in combination with TMZ/IR in patient-derived xenograft (PDX) models.